Biology subjects

Carvalho, A.

Publications and source records attributed to Carvalho, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Gfa, a new integron-borne fosfomycin resistance family

Fosfomycin is increasingly used to treat multidrug-resistant Gram-negative infections, yet resistance surveillance largely relies on homology-based detection of known fos genes. Here, we identify Gfa, a previously unrecognized integron-borne fosfomycin resistance determinant uncovered through functional screening of 129 integron gene cassettes. Gfa confers high-level fosfomycin resistance across Enterobacterales and Pseudomonas aeruginosa and is resistant to phosphonoformate inhibition. Structural modelling and mutagenesis reveal a conserved cysteine-rich, zinc-organized architecture essential for activity and distinct from canonical Fos enzymes. Crude-extract assays confirm glutathione-dependent fosfomycin inactivation under metal-chelating conditions. Distant Gfa homologs also conferred resistance, although activity was restricted to a subset of proteins tested. Together, these findings establish Gfa as a previously unrecognized fosfomycin resistance family and highlight the limitations of homology-based mining for discovering novel resistance determinants and predicting which family members are functional.

microbiology↗

bioRxiv2026

ANIMA: predicting protein-protein interactions across species

Motivation: Protein--protein interactions (PPIs) underpin a wide range of biological functions in living organisms. Experimental identification of new PPIs is expensive and time-consuming. The experimental bottleneck has implied an imbalance in terms of data availability: while certain species have been screened exhaustively, other species have not been sufficiently examined. An AI driven protocol for PPI prediction that leverages the massive data accumulated for certain species means a decisive boost for so far understudied species. Results: We present ANIMA (Artificial Neural Interaction Model for Animals), an AI supported cross-species PPI prediction model trained on popular species to predict PPIs in under-researched species. Our experiments demonstrate that our model, when trained on 200 diversely selected animal species, can successfully predict PPIs in other species: ANIMA achieves 95.3% accuracy on other animal species, 91.1% on other eukaryotes, and 82.5% on non-eukaryotes. For a more fine-grained evaluation of the model, we stratify performance rates by the evolutionary distance of test to training sets. We also stratify results by a novel, alignment based score ("representation score") which allows for fine-grained evaluation in terms of its capacity to generalize to unseen interactions. As expected, results demonstrate increasing performance on increasing evolutionary similarity and on increasing identity of interacting proteins, while still showing excellent performance on proteins entirely lacking counterparts in the training set. In comparison with the state of the art, ANIMA demonstrates substantial superiority in terms of performance rates.

bioinformatics↗

bioRxiv2026

Hydrogel confinement enables subcutaneous delivery of vesicant antibody-drug conjugates

Antibody-drug conjugates (ADCs) deliver cytotoxic payloads to tumors with antibody selectivity, yet all approved ADCs are administered by intravenous (IV) infusion despite a strong patient and clinical preference for subcutaneous (SC) delivery. SC administration would reduce treatment burden, but many ADC payloads are vesicants that cause tissue necrosis upon local release, a liability amplified, not mitigated, by the dispersion-enhancing excipients used for SC antibody formulations. We developed an injectable diacetyl-L-tartaric anhydride-functionalized chitosan hydrogel (TACT) that addresses this conflict by confining ADCs within a protective SC depot. TACT is compatible with clinically approved ADC formulations without drug-product modification and provides drug-to-antibody ratio (DAR)-dependent release kinetics that support a quantitative relationship with in vivo absorption timing. In direct comparison, recombinant human hyaluronidase (rHuPH20) co-formulated with vesicant ADCs caused severe tissue necrosis, whereas TACT prevented macroscopic injury while preserving antitumor efficacy comparable to intravenous dosing. TUNEL staining of injection sites showed that TACT attenuated peri-depot apoptotic injury 3-fold relative to T-DM1 alone and 2-fold relative to rHuPH20 co-formulation. In non-human primates, SC TACT achieved 78% relative bioavailability for total trastuzumab, reduced peak circulating T-DM1 catabolite (free DM1) exposure 7.6-fold compared to IV administration and produced only transient, self-resolving cutaneous reactions. These results identify depot-mediated confinement as a viable alternative to excipient-mediated dispersion for SC delivery of vesicant ADCs, demonstrated here for trastuzumab-based conjugates across two approved ADC drug products (T-DM1 and T-DXd, with non-cleavable MCC and cleavable peptide linkers), with supporting validation in a custom cleavable monomethyl auristatin E (MMAE) series. Additional validation with enfortumab vedotin (EV), a Nectin-4-targeting MMAE ADC, supported the applicability of this strategy beyond trastuzumab-based conjugates.

cancer biology↗

bioRxiv2026

Characterization of ovine follicular fluid and granulosa cell-derived extracellular vesicles and their miRNA cargo following in vitro exposure to bisphenols A and S.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

molecular biology↗

bioRxiv2026

Large-scale recovery of integron cassettes for gene discovery screens

Integrons capture and stockpile adaptive genes encoded in modular mobile genetic elements called integron cassettes (ICs). Present in 17% of bacterial genomes, integrons can harbor hundreds of cassettes, representing a hotspot of genetic variability. Some ICs encode antimicrobial or phage resistance genes, but most remain functionally uncharacterized, representing an untapped source of genes of biotechnological interest. Here, we present two tools, the cassette gatherer and hunter, that allow the swift establishment of libraries of genes either from genetically tractable strains or directly from DNA. By re-engineering a class 1 integron, these platforms capture single cassettes in a sequence and function-independent manner. When applied to Vibrio genomes, they recovered hundreds of single cassettes per assay with >99% specificity. To validate the usability of our tools for the discovery of new genes, we subjected these libraries to screens against phages ICP2 and T4, and identified nine phage-defense systems, including five previously undescribed. Hence, these tools provide a fast and simple method to recover thousands of ICs, in a way amenable to gene-discovery screens.

microbiology↗

bioRxiv2024

Metabolic reprogramming regulates histone lactylation during zebrafish caudal fin regeneration

Tissue regeneration relies on precise molecular mechanisms controlling cell fate transitions, with cell metabolism emerging as a key regulator. Lactate-derived histone lactylation has recently been identified as an important epigenetic modification involved in the regulation of gene expression across various biological processes. In this study, we report an increase in global histone lactylation and H3K18Lac levels in the mesenchyme and osteoblasts of the zebrafish caudal fin, during the early stages of regeneration. Our findings demonstrate that this epigenetic modification is functionally regulated by increased lactate levels, while inhibition of glycolysis and lactate production significantly reduces histone lactylation. These results suggest that metabolic reprogramming, in response to caudal fin injury, regulates histone lactylation, potentially modulating gene expression essential for cell plasticity and proliferation. This study expands our understanding of how metabolic-epigenetic interactions influence regenerative processes, providing valuable insights for the development of novel therapeutic strategies to enhance tissue repair. Summary statementLactate-derived histone lactylation plays a role in the early stages of zebrafish caudal fin regeneration, coupling metabolic adaptation with tissue repair mechanisms, through epigenetic regulation.

developmental biology↗

bioRxiv2024

Nitrosation of CD36 regulates endothelial function and serum lipids

During obesity, endothelial cells (ECs) become lipid laden leading to endothelial dysfunction. We demonstrate endothelium downregulates caveolin-1 (Cav1) in mouse and human in response to obesity. Using an EC-specific Cav1 knockout mouse, we find mice are hyperlipidemic regardless of diet, but retain endothelial cell function. Whereas initially this was thought to be due to Cav1 mediate endocytosis, we find instead the mice have significantly increased nitric oxide (NO) in response to the lack of Cav1. The presence or absence of NO toggled inversely EC lipid content and plasma lipid in mice. We found the fatty acid translocase CD36 was directly nitrosated by endogenous NO at the same cysteines that are palmitoylated on CD36. The nitrosation of CD36 prevented its trafficking to the plasma membrane and decreased lipid uptake. The physiological effect of this mechanism was a reliance on NO for endothelial function. This work suggests that CD36 nitrosation occurs as a protective mechanism to prevent EC lipotoxicity and preserve function. TeaserNitric oxide regulates serum lipids and endothelial cell lipid content through nitrosation of CD36.

molecular biology↗

bioRxiv2024

The expression of integron arrays is shaped by the translation rate of cassettes

Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the Pc promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show that the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, poorly translated cassettes decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward a novel integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the Pc.

microbiology↗

bioRxiv2024

Knowledge Innovation Ecosystem for the Promotion of User-Centre Health Innovations: Living Lab Methodology and Lessons Learned Through the Proposal of Standard Good Practices

Living Labs, experiencing a global surge in popularity over the past years, demands standardized guidance through the development of widely accepted good practices. While challenging due to the complex and evolving nature of Living Labs, this task remains essential. These knowledge innovation ecosystems facilitate a diverse array of interconnected and interacting end-users and stakeholder partners who engage collaboratively to co-create, embed, and/or leverage end-user-centric breakthroughs at one or more innovation phases within a real-world context. Based on the development of six Living Labs in the health domain, this study proposes a more general yet critical set of Living Labs good practices, emphasizing the importance of strong initial marketing and promotion strategies for Living Labs open calls, enforcing gender equality, carefully selecting stakeholders, devising and implementing effective framework strategies for end-user engagement and value creation, ensuring value creation for all Living Labs partners, prolonging the long-term viability of the Living Lab project, promoting and disseminating impactful actions and results, fostering environmental sustainability, and processing results data for Living Lab performance evaluation.

scientific communication and education↗

bioRxiv2023

Preserved striatal innervation and motor function despite severe loss of nigral dopamine neurons following mitochondrial dysfunction induced by mtDNA mutations

Degeneration of dopamine neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinsons disease (PD). In idiopathic cases, high levels of mitochondrial DNA (mtDNA) mutations associated with mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopamine neurons, leading to accelerated mtDNA ageing. K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although already [~]70% of nigral dopamine neurons had perished. The remaining neuron population still preserved [~]75% of axon terminals in the dorsal striatum, which enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of surviving nigral dopamine neurons. We conclude that a small population of substantia nigra neurons can adapt to mtDNA mutations and maintain motor control in mice, holding chances for new treatment strategies in PD patients.

neuroscience↗

bioRxiv2023

Gibberellin and the miRNA156-targeted SlSBPs synergistically regulate tomato floral meristem activity and fruit patterning

Many developmental processes associated with fruit development take place at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GA) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, we show that GA and miR156 function in tomato FM and fruit patterning. High GA responses or overexpression of miR156 (156OE), which leads to low levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN- LIKE (SPL/SBP), resulted in enlarged FMs, defects in FM determinacy and fruits with increased locule number. Conversely, low GA responses reduced fruit indeterminacy and locule number, and overexpression of a miR156-resistant SlSBP15 allele (rSBP15) reduced cell number and size in the FM, as well as locule number. GA responses were partially required for the fruit defects observed in 156OE and rSBP15 plants. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBPs, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to genetically fine-tune aspects of fruit development that have been important for tomato domestication.

plant biology↗

bioRxiv2023

Ghrelin delays premature aging in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition arising from a single nucleotide alteration in the LMNA gene, which leads to the production of a defective lamin A protein known as progerin. The buildup of progerin hastens the onset of premature and expedited aging. Patients with HGPS exhibit short stature, low body weight, lipodystrophy, metabolic dysfunction, and skin and musculoskeletal abnormalities and, in most cases, die of cardiovascular disease by their early teenage years. Currently, no effective cure or treatment for the disease highlights the importance of discovering new therapeutic strategies. Herein, we present evidence that the hormone ghrelin, besides promoting autophagy and progerin clearance, rescued several cellular hallmarks of premature aging of human HGPS fibroblasts. Using an HGPS mouse model, LmnaG609G/G609G mice, we also show that ghrelin administration rescued the short-lived mice molecular and histopathological progeroid features, prevented progressive weight loss at later stages, reverted the lipodystrophic phenotype, and extended lifespan. Thus, we disclose that modulation of ghrelin signaling may give rise to new treatment targets and translational approaches that may improve outcomes and the health quality of HGPS patients and natural aging pathologies.

physiology↗

bioRxiv2023

RavA-ViaA links Vibrio cholerae Cpx and Zra2 envelope stress to antibiotic response

RavA-ViaA were reported to play a role in aminoglycoside sensitivity but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high aminoglycoside concentrations, during aerobic growth. Using high throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the aminoglycoside tolerance of{Delta} ravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response aminoglycosides because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on polymyxin B sensitivity and vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug resistant Gram-negative bacteria.

microbiology↗

bioRxiv2022

Queuosine modification of tRNA-Tyrosine elicits translational reprogramming and enhances growth of Vibrio cholerae with aminoglycosides.

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q34 in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overall bacterial proteome, and elucidated the molecular mechanisms underlying the effects of Q34 modification in antibiotic translational stress response. Using molecular reporters, we showed that Q34 impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics analyses revealed that the anti-SoxR factor RsxA is better translated in the absence of tgt. RsxA displays a codon bias towards tyrosine TAT and overabundance of RsxA leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identified conditions that regulate tgt expression. We propose that regulation of Q34 modification in response to environmental cues leads to translational reprogramming of transcripts bearing a biased tyrosine codon usage. In silico analysis further identified candidate genes which could be subject to such translational regulation, among which DNA repair factors. Such transcripts, fitting the definition of modification tunable transcripts, are central in the bacterial response to antibiotics.

microbiology↗

bioRxiv2022

Re-direction of phagosomes to the recycling expulsion pathway by a fungal pathogen

The analysis of host-pathogen interactions bears the potential to discover novel pathogenicity mechanisms and to obtain novel insights into basic mechanisms of cell biology. Here, we obtained unprecedented insight into both. We discovered that the HscA protein on the conidial surface of the clinically important human-pathogenic fungus Aspergillus fumigatus acts as an effector protein. It inhibits phagosome maturation and reprograms phagosomes for expulsion of conidia. HscA anchors the human p11 protein to phagosomes. p11 is a decisive factor for targeting phagosomes either to the degradative or secretory pathway. The relevance of our findings is indicated by the identification of an SNP in the non-coding region of the human p11 gene that affects its translation and is associated with heightened susceptibility to invasive pulmonary aspergillosis.

cell biology↗

bioRxiv2022

miR319-targeted TCP4/LANCEOLATE directly regulates OVATE and auxin responses to modulate tomato gynoecium patterning and fruit morphology

Diversity in fruit morphology is one of the hallmarks of varietal differences among modern cultivars of fruit-bearing crops. As evolutionarily related organs, fruits and leaves share developmental processes, but there are surprisingly few connections between regulatory pathways for fruit and leaf development. Here, we show the regulation of the leaf development-associated TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP4/LANCEOLATE (TCP4/LA) transcription factor by the microRNA319 (miR319) is crucial for gynoecium patterning and establishment of fruit morphology. Loss of miR319 regulation leads to a premature, ectopic TCP4/LA expression during gynoecium patterning, which results in elongated fruits, resembling ovate mutants. TCP4/LA modulates tomato fruit development and morphology partially by directly repressing OVATE expression as early as 5-8 days post-inflorescence (dpi) flower buds. Furthermore, miR319-targeted CINCINNATA-like TCP4/LANCEOLATE controls auxin responses in developing flower buds by directly binding to the SlYUCCA4 promoter. Modulation of auxin biosynthesis by TCP4/LA is shared with other CINCINNATA-like TCPs during Arabidopsis gynoecium patterning. Our study defines a novel miRNA-based molecular link between OVATE, a fundamental gene associated with tomato domestication, and auxin responses in the control of fruit development and morphology. Given the striking variation in fruit shape among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of hormone dynamics may be a common driver in the evolution of fruit-shape diversity.

plant biology↗

bioRxiv2022

A microfluidics-assisted methodology for label-free isolation of CTCs with downstream methylation analysis of cfDNA in lung cancer

Lung cancer (LC) is a major cause of mortality. Late diagnosis, associated with limitations in tissue biopsies for adequate tumor characterization contribute to limited survival of lung cancer patients. Liquid biopsies have been introduced to improve tumor characterization through the analysis of biomarkers, including circulating tumour cells (CTCs) and cell-free DNA (cfDNA). Considering their availability in blood, several enrichment strategies have been developed to augment circulating biomarkers for improving diagnostic, prognostic and treatment efficacy assessment; often, however, only one biomarker is tested. In this work we developed and implemented a microfluidic chip for label-free enrichment of CTCs with a methodology for subsequent cfDNA analysis from the same cryopreserved sample. CTCs were successfully isolated in 38 of 42 LC patients with the microfluidic chip. CTCs frequency was significantly higher in LC patients with advanced disease. A cut-off of 1 CTC/mL was established for diagnosis (sensitivity=76.19%, specificity=100%) and in patients with late-stage lung cancer, the presence of [≥] 5 CTCs/mL was significantly associated with shorter overall survival. MIR129-2me and ADCY4me panel of cfDNA methylation performed well for LC detection, whereas MIR129-2me combined with HOXA11me allowed for patient risk stratification. Analysis of combinations of biomarkers enabled the definition of panels for LC diagnosis and prognosis. Overall, this study demonstrates that multimodal analysis of tumour biomarkers via microfluidic devices may significantly improve LC characterization in cryopreserved samples, constituting a reliable source for continuous disease monitoring.

bioengineering↗

bioRxiv2022

Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress

Antimicrobial resistance (AMR) develops as a major problem in infectious diseases treatment. While antibiotic resistance mechanisms are usually studied using lethal antibiotic doses, lower doses allowing bacterial growth are now considered as factors influencing the development and selection of resistance. Based on high throughput transposon insertion sequencing (TN-seq) in V. cholerae, we have undertaken the phenotypic characterization of 23 transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications deletion mutants, for which growth is globally not affected in the absence of stress. We uncover a specific involvement of different RNA modification genes in the response to aminoglycosides (tobramycin (TOB), gentamicin (GEN)), fluoroquinolones (ciprofloxacin (CIP)), {beta}-lactams (carbenicillin (CRB)), chloramphenicol (CM) and trimethoprim (TRM). Our results identify t/rRNA modification genes, not previously associated to any antibiotic resistance phenotype, as important factors affecting the bacterial response to sub-MIC antibiotics from different families. This suggests differential translation and codon decoding as critical factors involved in the bacterial response to stress.

microbiology↗